Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32]orthophosphoric acid, they synthesized [32]prothymosin alpha. Thin layer electrophoresis of partially hydrolyzed labeled protein indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine and [32]human prothymosin alpha revealed that a 14-mer derived from the amino terminus was phosphorylated at a single position despite the presence of three closely spaced serine residues. Approximately 2% of the peptide in each case contained phosphate. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only on the N- terminal acetylserine residue. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not govern mitosis. Prothymosin alpha pre-mRNAs are alternatively spliced as a consequence of adjacent AG acceptor couplets (GAGGAG): at the intron 2/exon 3 boundary of the only expressed human prothymosin alpha gene. In all human cells and tissues examined, two mRNA transcripts in the ratio of 9:1, shorter form: longer form were observed. Production of the shorter mRNA, which utilizes the second AG dinucleotide, violates two consensus rules for splice site selection. The poor performance of the first AG dinucleotide cannot be explained by its position relative to other splicing signals, judging by a study or mutant genes. Tie GAGGAG motif in the prothymosin alpha gene has been retained by the African monkey, Colobus, suggesting that the ambiguity in splice-site selection confers a selective advantage. A new, simple and highly efficient method for generating mutants called Phoenix Mutagenesis has been devised.